This is an ongoing comprehensive program for study at the molecular level of composition and mechanistic details of mitochondrial electron transport; energy conservation, transduction and transfer; ATP synthesis and hydrolysis. Five enzyme complexes have been isolated: I, II, III and IV are segments of the respiratory chain and capable of total respiratory activity reconstitution; V catalyzes oligomycin- and uncoupler-sensitive ATP-Pi exchange at 350-470 nmoles times min-1 times mg-1protein, and contains the mitochondrial uncoupler-binding protein as determined by equilibrium binding and photoaffinity labeling studies using radioactive 2-azido-4-nitrophenol. The purpose of the proposed research is in complex I: to study mechanisms of NADH and NADPH oxidation and ubiquinone (Q) reduction, isolate and purify the iron-sulfur proteins bearing centers 1, 2, 3, 4, study mechanisms of energy conservation and transfer; in complex II: to study mechanism of Q reduction, role of cytochrome b557.5 and the low-potential iron-sulfur center S-2, purify b557.5; in complex III: Purify cytochromes bK and bT, study their regulatory properties study and better characterize chromophore-558, study mechanism of energy conservations and transfer, components required for these functions, the reason for apparent absence of ATP-Pi exchange, whether vesicle formation is required for ATP-Pi exchange, the role and possible relevance to energy conservation-transfer of the new FAD containing iron-sulfur flavoprotein which is tightly associated with complex V, reconstitution of V with I, III and IV for energy transfer and oxidative phosphorylation. Also, work is in progress on protein factors required for mitochondrial energy-linked functions. Our current thinking is that these factors are involved in energy transfer among complexes, I, III, IV and V as they appear to be required for energy-linked functions, but not for electron transfer, ATP hydrolysis, proton translocation and maintenance of transmembrane proton gradient.